Project Summary/ Abstract The human immunodeficiency virus (HIV) continues to elude eradication. While combination antiretroviral therapy (cART) is effective in decreasing HIV-associated morbidity and mortality, infected individuals face a lifetime of treatment and myriad potential complications including end organ diseases such as HIV-associated neurocognitive disorder (HAND). Growing evidence shows that the central nervous system (CNS) is one of the viral reservoir sites that enables the virus to evade eradication. Specifically, the myeloid cell lineage represents the primary immune cell of the CNS and there is evidence that this cell lineage could harbor HIV. With the discovery of meningeal lymphatics, it is also possible that lymphocyte populations of the CNS may also harbor HIV. However, more evidence is needed from live humans to better understand these reservoirs, including its size and its characteristics that may differ from the T- lymphocyte reservoir. Our team of Emory/CDC/UCSD investigators will perform an innovative investigation with two major aims to address the HIV immune cell reservoir in the CNS. First, we will leverage archived plasma/cerebrospinal fluid (CSF) samples collected in UCSD/Emory studies from individuals with significant HIV RNA levels in both compartments. With our group members at CDC, we will test the virus in these samples for association with either myeloid or T-lymphocyte cell surface markers using an innovative immuncapture technique. This will determine the relative HIV contributions from each cell type in the CSF. Virions will then be sequenced using traditional approaches as well as a new CDC approach (MicrobeTRACE) that provides a three dimensional picture of genetic relatedness. Second, we will conduct a prospective study of HIV+ individuals on cART to further characterize plasma/CSF myeloid and T-lymphocyte cell populations that we have identified in preliminary work. We will also examine for the presence of HIV in these cells as well as differences in HIV genetic sequences between cells. Results from the two aims will be interpreted in the setting of other biomarkers as well as neuropsychological performance. We are confident that the investigation will bring the field closer to a full understanding of the HIV CNS reservoir and could provide a roadmap for HIV eradication therapies targeting both myeloid and T-lymphocyte populations in the CNS.